Project Summary Glioblastoma multiforme (GBM) is the most common form of malignant primary brain tumor and is one of the most lethal cancers. Despite the academic and industrial efforts to find better treatment, the 5-year survival of GBM patients has not changed over the past 10 years and is less than 6%. GBM is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches, most of which are based on apoptosis induction in cancer cells. Over the past decade, a number of cell death pathways based on alternative non-apoptotic mechanisms have been discovered. The PI's research team has recently identified the fungal secondary metabolite ophiobolin A (OpA) as an agent with significant activity against apoptosis-resistant GBM cells through the induction of paraptotic cell death. Paraptosis is a recently discovered form of non-apoptotic cell death, which is distinguished from apoptosis by the absence of apoptotic morphology, DNA fragmentation or caspase activation. Paraptosis was found to be clinically relevant based on transmission electron microscopy analysis of human brain tumor tissue, which revealed paraptotic characteristics in GBM cells undergoing cell death. The discovery of OpA as a paraptotic agent demonstrates for the first time the possibility of paraptosis induction in GBM cells with a small molecule and lays the foundation to explore paraptosis as an innovative strategy to combat GBM. However, the OpA's mode of action is likely based on covalent modification of its intracellular target(s) and thus possible off-target reactivity needs to be addressed. This application involves the investigation of an acid-sensitive OpA prodrug approach that will exploit the elevated acidity of the GBM microenvironment to enhance the selectivity for tumor targeting. The proposed prodrug approach will also allow for the targeting of glioma stem cells (GSCs), which are considered to be responsible for the ineffectiveness of current GBM therapeutic agents. The proposed work is based on the preliminary results involving the discovery of OpA C5 and C21 bis-acetals that can be efficiently synthesized and cleanly hydrolysed under acid catalysis to regenerate the parent drug. The project is aimed at exploiting this discovery to identify prodrugs that will undergo hydrolysis at tumor relevant pH of 6.2 and show high selectivity at killing GBM cells grown in cultures at reduced pH compared to those maintained under normal neutral conditions. Compounds possessing such characteristics will be subjected to pharmacokinetics and toxicity/efficacy studies in a mouse model of human GBM using OpA as a control. These studies are expected to facilitate the development of OpA (or its superior analogue) as a first-in-class pro-paraptotic anti-GBM agent by investigating its potential use in a prodrug form and ultimately aim to establish the concept of paraptosis as a new strategy for GBM therapy.